Retroviruses co-opt cellular pathways to facilitate the assembly of virus particles that spread infection. Identifying the host factors usurped by retroviruses for virus assembly will enhance our understanding of retrovirus-cell interactions and may uncover new targets for antiviral therapy. Although host factors engaged in late steps of retrovirus assembly and release have been identified, the early stages of assembly remain poorly understood because the molecular events that initiate assembly have been difficult to study. However, our discovery that the Rous sarcoma virus (RSV) Gag protein, which orchestrates the assembly process, undergoes nuclear trafficking has allowed us to study early events in the assembly pathway that occur in the nucleus. Using this system, we showed that nuclear trafficking of RSV Gag is crucial for the formation of infectious virus particles, and mutants that bypass the nucleus incorporate reduced amounts of viral genomic RNA. Furthermore, our structural studies demonstrate that Gag binding to nuclear export machinery is facilitated by Gag-RNA interactions, providing a mechanism for the regulated transport of viral ribonucleoprotein complexes. New data from our laboratory suggest that RSV Gag may traffic to sites of active transcription to recognize and select viral genomic RNA in the nucleus. Therefore, our research may define a novel paradigm for how retroviruses select and package their genomes. In this application, we propose two Specific Aims to test the novel hypothesis that Gag nuclear trafficking facilitates recognition, selection, and packaging of the genomic RNA. These mechanistic experiments take advantage of our well-characterized toolbox of Gag mutants with altered nuclear trafficking and RNA packaging properties. We will utilize our unique set of reagents to investigate whether Gag is tethered near sites of RNA synthesis to mediate co-transcriptional packaging of viral genomic RNA. In addition, we will use mass spectrometry to identify host factors that interact with RSV Gag and deep sequencing to analyze cellular RNA targets of Gag. Proteomic analysis of Gag-interacting factors from diverse retroviruses that undergo nuclear localization will broaden the impact of this application and provide insight into potential common functions of retroviral Gag proteins in the nucleus. Through this well-crafted, focused experimental plan, we will shed light on fundamental aspects of retrovirus assembly, genomic RNA packaging, and nuclear events to reveal new information about the interplay between retroviral pathogens and their host cells.